Multiple CpG oligodeoxynucleotides and their use to induce an immune response

ABSTRACT

Compositions including multiple oligodeoxynucleotides with a CpG motif are disclosed herein. The compositions can include either D or K type oligodeoxynucleotides. These compositions are of use in inducing an immune response in a large percentage of the individuals in a population.

PRIORITY CLAIM

This is a divisional application of U.S. patent application Ser. No.10/194,035, filed on Jul. 12, 2002, now issued U.S. Pat. No. 7,521,063,which is a continuation-in-part of PCT Application No. PCT/US01/01122filed on Jan. 12, 2001, which claims the benefit of U.S. ProvisionalPatent Application No. 60/176,115 filed on Jan. 14, 2000, all of whichare incorporated herein by reference in their entirety.

FIELD

This application relates to oligodeoxynucleotides including a CpG motif,specifically to the use of multiple oligodeoxynucleotides including aCpG motif for inducing an immune response.

BACKGROUND

DNA is a complex macromolecule whose immunological activities areinfluenced by its base composition and base modification, as well ashelical orientation. Certain unusual DNA structures (e.g., Z-DNA) caninduce significant antibody responses when administered to normal mice.In addition, bacterial DNA, as well as certain synthetic unmethylatedCpG sequences can induce proliferation and immunoglobulin (Ig)production by murine B cells. Unmethylated CpG dinucleotides are morefrequent in the genomes of bacteria and viruses than vertebrates. Recentstudies suggest that immune recognition of these motifs may contributeto the host's innate immune response. D. M. Klinman et al., “CpG MotifsPresent in Bacterial DNA Rapidly Induce Lymphocytes to SecreteInterleukin 6, Interleukin 12, and Interferon γ,” 93 Proc. Natl. Acad.Sci. USA 2879 (1996); A.-K. Yi et al., “Rapid Immune Activation by CpGMotifs in Bacterial DNA,” 157 J. Immun. 5394 (1996); Hua Liang et al.,“Activation of Human B Cells by Phosphorothioate Oligodeoxynucleotides,”98 J. Clin. Invest. 1119 (1996); A. M. Krieg et al., “CpG Motifs inBacterial DNA Trigger Direct B-Cell Activation,” 374 Nature 546 (1995).

In mice, CpG DNA induces proliferation in almost all (>95%) of B cellsand increases Ig secretion. This B cell activation by CpG DNA is T cellindependent and antigen non-specific. In addition to its direct effectson B cells, CpG DNA also directly activates monocytes, macrophages, anddendritic cells to secrete a variety of cytokines. These cytokinesstimulate natural killer (NK) cells to secrete γ-interferon (IFN-γ) andhave increased lytic activity. Examples of which can be found inInternational Patent Applications WO 95/26204, WO 96/02555, WO 98/11211,WO 98/18810, WO 98/37919, WO 98/40100, WO 98/52581, and PCT/US98/047703;U.S. patent application Ser. Nos. 08/738,652 and 09/136,138; and U.S.Pat. No. 5,663,153.

Although bacterial DNA and certain CpG sequences can induce responsesfrom human cells (Ballas et al., “Induction of NK Activity in Murine andHuman Cells by CpG Motifs in Oligodeoxynucleotides and Bacterial DNA,”157 J. Immunol. 1840 (1996)), individual subjects show considerableheterogeneity in their response to different CpG sequences. As disclosedherein, CpG sequences that strongly stimulate cells from some subjectsare virtually inactive on cells from other subjects. These differentresponses can make it difficult to induce a therapeutic immune responsein all members of a diverse population using a single CpG sequence, evenif such a sequence is expressed repetitively in a givenoligodeoxynucleotide. Thus, there exists a need to identify differentCpG sequences that together are capable of optimally inducing an immuneresponse in cells from all members of a target population.

SUMMARY

Compositions including multiple oligodeoxynucleotides with a CpG motifare disclosed herein. These compositions are of use in inducing animmune response in a large percentage of the individuals in apopulation.

In one embodiment, an oligodeoxynucleotide composition is disclosed thatincludes at least two oligodeoxynucleotides of at least about 10nucleotides in length, wherein the oligodeoxynucleotides each include anunmethylated CpG motif. In this composition, the oligodeoxynucleotideshave a sequence represented by the formula 5′ N₁N₂N₃Q-CpG-WN₄N₅N₆ 3′,wherein Q is T or G or A, W is A or T, and N₁, N₂, N₃, N₄, N₅, and N₆are any nucleotide. The two oligodeoxynucleotides differ in theirnucleic acid sequence. In one embodiment, the oligodeoxynucleotidecompositions are used to induce an immune response. In one specific,non-limiting example, the oligodeoxynucleotide compositions are used toinduce an immune response in at least about 80% of a population. In onespecific, non-limiting example, a single parameter of the immuneresponse can be increased. In another specific, non-limiting example,administration of the at least two oligodeoxynucleotides broadens theimmune response. For example, more parameters of the immune response areincreased by administering the at least two oligodeoxynucleotidesincluding a CpG motif, than are stimulated upon administration of onlyone of the oligodeoxynucleotides including a CpG motif.

In another embodiment, an oligodeoxynucleotide composition is disclosedthat includes at least two oligodeoxynucleotides of at least about 10nucleotides in length, wherein the oligodeoxynucleotides comprise anunmethylated CpG motif, wherein the oligodeoxynucleotides include asequence represented by the formula 5′ RY-CpG-RY 3′, wherein R is A or Gand Y is C or T. The at least two oligodeoxynucleotides differ in theirnucleic acid sequence. In one embodiment, the oligodeoxynucleotidecompositions are used to induce an immune response. In one specific,non-limiting example, the oligodeoxynucleotide compositions are used toinduce an immune response in at least about 80% of a population. In onespecific, non-limiting example, a single parameter of the immuneresponse can be increased. In another specific, non-limiting example,administration of the at least two oligodeoxynucleotides broadens theimmune response. Thus, more parameters of the immune response areincreased by administering the at least two oligodeoxynucleotides thatinclude a CpG motif, than are stimulated upon administration of only oneof the oligodeoxynucleotides that includes a CpG motif.

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822.

DETAILED DESCRIPTION I. Abbreviations

A: adenine

Ab: antibody

APC: antigen presenting cell

C: cytosine

APC: antigen presenting cell

CpG ODN: an oligodeoxynucleotide (either a D or a K type) including aCpG motif.

DC: dendritic cell

FCS: fetal calf serum

G: guanine

h: hour

HKLV: heat-killed leishmania vaccine

IFN-α: interferon alpha

IFN-γ: interferon gamma

IL-10: interleukin 10

IL-6: interleukin 6

mm: millimeter

mRNA: messenger ribonucleic acid.

ODN: oligodeoxynucleotide

Pu: purine

Py: pyrimidine

s.c.: subcutaneous

T: thymine

μg: microgram

II. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of theinvention, the following explanations of specific terms are provided:

Allergen: A substance that can induce an allergic or asthmatic responsein a susceptible subject. The list of allergens is enormous and caninclude pollens, insect venoms, animal dander dust, fungal spores anddrugs (e.g. penicillin). Examples of natural, animal and plant allergensinclude proteins specific to the following genera: Canine (Canisfamiliaris); Dermatophagoides (e.g. Dermatophagoides farinae); Felis(Felis domesticus); Ambrosia (Ambrosia artemiisfolia); Lolium (e.g.Lolium perenne or Lolium multiflorum); Cryptomeria (Cryptomeriajaponica); Alternaria (Alternaria alternata); Alder; Alnus (Alnusgultinosa); Betula (Betula verrucosa); Quercus (Quercus alba); Olea(Olea europa); Artemisia (Artemisia vulgaris); Plantago (e.g. Plantagolanceolata); Parietaria (e.g. Parietaria officinalis or Parietariajudaica); Blattella (e.g. Blattella germanica); Apis (e.g. Apismultiflorum); Cupressus (e.g. Cupressus sempervirens, Cupressusarizonica and Cupressus macrocarpa); Juniperus (e.g. Juniperussabinoides, Juniperus virginiana, Juniperus communis and Juniperusashei); Thuya (e.g. Thuya orientalis); Chamaecyparis (e.g. Chamaecyparisobtusa); Periplaneta (e.g. Periplaneta americana); Agropyron (e.g.Agropyron repens); Secale (e.g. Secale cereale); Triticum (e.g. Triticumaestivum); Dactylis (e.g. Dactylis glomerata); Festuca (e.g. Festucaelatior); Poa (e.g. Poa pratensis or Poa compressa); Avena (e.g. Avenasativa); Holcus (e.g. Holcus lanatus); Anthoxanthum (e.g. Anthoxanthumodoratum); Arrhenatherum (e.g. Arrhenatherum elatius); Agrostis (e.g.Agrostis alba); Phleum (e.g. Phleum pratense); Phalaris (e.g. Phalarisarundinacea); Paspalum (e.g. Paspalum notatum); Sorghum (e.g. Sorghumhalepensis); and Bromus (e.g. Bromus inermis). The term “allergy” refersto acquired hypersensitivity to a substance (allergen). An “allergicreaction” is the response of an immune system to an allergen in asubject allergic to the allergen. Allergic conditions include eczema,allergic rhinitis or coryza, hay fever, bronchial asthma, urticaria(hives) and food allergies, and other atopic conditions.

Antigen: A compound, composition, or substance that can stimulate theproduction of antibodies or a T-cell response in an animal, includingcompositions that are injected or absorbed into an animal. An antigenreacts with the products of specific humoral or cellular immunity,including those induced by heterologous immunogens. The term “antigen”includes all related antigenic epitopes.

Anti-infectious agent: A substance (such as a chemical compound,protein, antisense oligonucleotide, or other molecule) of use intreating infection of a subject. Anti-infectious agents include, but arenot limited to, anti-fungals, anti-virals, and antibiotics.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects.

Asthma: A disorder of the respiratory system characterized byinflammation, narrowing of the airways and increased reactivity of theairways to inhaled agents. Asthma is frequently, although notexclusively associated with atopic or allergic symptoms.

CpG or CpG motif: A nucleic acid having a cytosine followed by a guaninelinked by a phosphate bond in which the pyrimidine ring of the cytosineis unmethylated. The term “methylated CpG” refers to the methylation ofthe cytosine on the pyrimidine ring, usually occurring at the 5-positionof the pyrimidine ring. A CpG motif is a pattern of bases that includean unmethylated central CpG surrounded by at least one base flanking (onthe 3′ and the 5′ side of) the central CpG. Without being bound bytheory, the bases flanking the CpG confer part of the activity to theCpG oligodeoxynucleotide. A CpG oligodeoxynucleotide is anoligodeoxynucleotide that is at least about ten nucleotides in lengthand includes an unmethylated CpG. CpG oligodeoxynucleotides include bothD and K type oligodeoxynucleotides (see below). CpGoligodeoxynucleotides are single-stranded. The entire CpGoligodeoxynucleotide can be unmethylated or portions may beunmethylated. In one embodiment, at least the C of the 5′ CG 3′ isunmethylated.

Cancer: A malignant neoplasm that has undergone characteristic anaplasiawith loss of differentiation, increase rate of growth, invasion ofsurrounding tissue, and is capable of metastasis. For example, thyroidcancer is a malignant neoplasm that arises in or from thyroid tissue,and breast cancer is a malignant neoplasm that arises in or from breasttissue (such as a ductal carcinoma). Residual cancer is cancer thatremains in a subject after any form of treatment given to the subject toreduce or eradicate thyroid cancer. Metastatic cancer is a cancer at oneor more sites in the body other than the site of origin of the original(primary) cancer from which the metastatic cancer is derived.

Chemotherapy; chemotherapeutic agents: As used herein, any chemicalagent with therapeutic usefulness in the treatment of diseasescharacterized by abnormal cell growth. Such diseases include tumors,neoplasms, and cancer as well as diseases characterized by hyperplasticgrowth such as psoriasis. In one embodiment, a chemotherapeutic agent isan agent of use in treating neoplasms such as solid tumors. In oneembodiment, a chemotherapeutic agent is radioactive molecule. One ofskill in the art can readily identify a chemotherapeutic agent of use(e.g. see Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 inHarrison's Principles of Internal Medicine, 14th edition; Perry et al.,Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2^(nd) ed., ©2000Churchill Livingstone, Inc; Baltzer L, Berkery R (eds): Oncology PocketGuide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; FischerD S, Knobf M F, Durivage H J (eds): The Cancer Chemotherapy Handbook,4th ed. St. Louis, Mosby-Year Book, 1993).

Cytokine: Proteins made by cells that affect the behavior of othercells, such as lymphocytes. In one embodiment, a cytokine is achemokine, a molecule that affects cellular trafficking. Specificnon-limiting examples of cytokines are IFN-γ, IL-6, and IL-10.

D Type Oligodeoxynucleotide (D ODN): An oligodeoxynucleotide includingan unmethylated CpG motif that has a sequence represented by theformula:5′RY-CpG-RY3′

wherein the central CpG motif is unmethylated, R is A or G (a purine),and Y is C or T (a pyrimidine). D-type oligodeoxynucleotides include anunmethylated CpG dinucleotide. Inversion, replacement or methylation ofthe CpG reduces or abrogates the activity of the D oligodeoxynucleotide.

In one embodiment, a D type ODN is at least about 16 nucleotides inlength and includes a sequence represented by Formula III:5′X₁X₂X₃Pu₁Py₂CpGPu₃Py₄X₄X₅X₆(W)_(M)(G)_(N)-3′

wherein the central CpG motif is unmethylated, Pu is a purinenucleotide, Py is a pyrimidine nucleotide, X and W are any nucleotide, Mis any integer from 0 to 10, and N is any integer from 4 to 10.Additional detailed description of D ODN sequences and their activitiescan be found in the section entitled “Multiple D and K TypeOligodeoxynucleotides (ODN) that Include a CpG Motif.” Generally D ODNscan stimulate a cellular response. For example, D ODNs stimulate naturalkiller cells and the maturation of dendritic cells.

Dendritic cell (DC): Dendritic cells are the principle antigenpresenting cells (APCs) involved in primary immune responses. Theirmajor function is to obtain antigen in tissues, migrate to lymphoidorgans and present the antigen in order to activate T cells.

When an appropriate maturational cue is received, DC are signaled toundergo rapid morphological and physiological changes that facilitatethe initiation and development of immune responses. Among these are theup-regulation of molecules involved in antigen presentation; productionof pro-inflammatory cytokines, including IL-12, key to the generation ofTh1 responses; and secretion of chemokines that help to drivedifferentiation, expansion, and migration of surrounding naive Th cells.Collectively, these up-regulated molecules facilitate the ability of DCto coordinate the activation and effector function of other surroundinglymphocytes that ultimately provide protection for the host. Althoughthe process of DC maturation is commonly associated with events thatlead to the generation of adaptive immunity, many stimuli derived fromthe innate branch of the immune system are also capable of activating DCto initiate this process. In this manner, DC provide a link between thetwo branches of the immune response, in which their initial activationduring the innate response can influence both the nature and magnitudeof the ensuing adaptive response. A dendritic cell precursor is a cellthat matures into an antigen presenting dendritic cell. In oneembodiment, a dendritic cell is a plasmacytoid dendritic cell.

Differentiation: The process by which cells become more specialized toperform biological functions, and differentiation is a property that istotally or partially lost by cells that have undergone malignanttransformation. For example, dendritic cell precursors undergomaturation to become APCs.

Epitope: An antigenic determinant. These are particular chemical groupsor peptide sequences on a molecule that are antigenic, i.e. that elicita specific immune response. An antibody binds a particular antigenicepitope.

Functionally Equivalent Sequence alterations, for example in a D typeODN, that yield the same results as described herein. Such sequencealterations can include, but are not limited to, deletions, basemodifications, mutations, labeling, and insertions.

Immune response: A response of a cell of the immune system, such as a Bcell, T cell to a stimulus. In one embodiment, the response is specificfor a particular antigen (an “antigen-specific response”). A “parameterof an immune response” is any particular measurable aspect of an immuneresponse, including, but not limited to, cytokine secretion (IL-6,IL-10, IFN-γ, etc.), immunoglobulin production, dendritic cellmaturation, and proliferation of a cell of the immune system. One ofskill in the art can readily determine an increase in any one of theseparameters, using known laboratory assays. In one specific non-limitingexample, to assess cell proliferation, incorporation of ³H-thymidine canbe assessed. A “substantial” increase in a parameter of the immuneresponse is a significant increase in this parameter as compared to acontrol. Specific, non-limiting examples of a substantial increase areat least about a 50% increase, at least about a 75% increase, at leastabout a 90% increase, at least about a 100% increase, at least about a200% increase, at least about a 300% increase, and at least about a 500%increase. One of skill in the art can readily identify a significantincrease using known statistical methods. One, specific, non-limitingexample of a statistical test used to assess a substantial increase isthe use of a Z test to compare the percent of samples that respond tomultiple ODNs including a CpG motif as compared to the percent ofsamples that respond using a single ODN including a CpG motif. Anon-paramentric ANOVA can be used to compare differences in themagnitude of the response induced by multiple ODNs including a CpG motifas compared to the percent of samples that respond using a single ODN.In this example, p≦0.05 is significant, and indicates a substantialincrease in the parameter of the immune response. One of skill in theart can readily identify other statistical assays of use.

Immune system deficiency: A disease or disorder in which the subject'simmune system is not functioning in normal capacity or in which it wouldbe useful to boost a subject's immune response. Immune systemdeficiencies include those diseases or disorders in which the immunesystem is not functioning at normal capacity, or in which it would beuseful to boost the immune system response. In one specific,non-limiting example, a subject with an immune system deficiency has atumor or cancer (e.g. tumors of the brain, lung (e.g. small cell andnon-small cell), ovary, breast, prostate, colon, as well as othercarcinomas and sarcomas).

Infectious agent: An agent that can infect a subject, including, but notlimited to, viruses, bacteria, and fungi.

Examples of infectious virus include: Retroviridae; Picornaviridae (forexample, polio viruses, hepatitis A virus; enteroviruses, humancoxsackie viruses, rhinoviruses, echoviruses); Calciviridae (such asstrains that cause gastroenteritis); Togaviridae (for example, equineencephalitis viruses, rubella viruses); Flaviridae (for example, dengueviruses, encephalitis viruses, yellow fever viruses); Coronaviridae (forexample, coronaviruses); Rhabdoviridae (for example, vesicularstomatitis viruses, rabies viruses); Filoviridae (for example, ebolaviruses); Paramyxoviridae (for example, parainfluenza viruses, mumpsvirus, measles Virus, respiratory syncytial virus); Orthomyxoviridae(for example, influenza viruses); Bungaviridae (for example, Hantaanviruses, bunga viruses, phleboviruses and Nairo viruses); Arena viridae(hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbivirusesand rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus);Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses, polyomaviruses); Adenoviridae (most adenoviruses); Herpesviridae (herpessimplex virus (HSV) 1 and HSV-2, varicella zoster virus, cytomegalovirus(CMV), herpes viruses); Poxviridae (variola viruses, vaccinia viruses,pox viruses); and Iridoviridae (such as African swine fever virus); andunclassified viruses (for example, the etiological agents of Spongiformencephalopathies, the agent of delta hepatitis (thought to be adefective satellite of hepatitis B virus), the agents of non-A, non-Bhepatitis (class 1=internally transmitted; class 2=parenterallytransmitted (i.e., Hepatitis C); Norwalk and related viruses, andastroviruses).

Examples of infectious bacteria include: Helicobacter pyloris, Boreliaburgdorferi, Legionella pneumophilia, Mycobacteria sps (such as M.tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae),Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis,Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus),Streptococcus agalactiae (Group B Streptococcus), Streptococcus(viridans group), Streptococcus faecalis, Streptococcus bovis,Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenicCampylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillusantracis, corynebacterium diphtheriae, corynebacterium sp.,Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridiumtetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturellamultocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillusmoniliformis, Treponema pallidium, Treponema pertenue, Leptospira, andActinomyces israelli.

Examples of infectious fungi include, but are not limited to,Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis,Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans.

Other infectious organisms (such as protists) include: Plasmodiumfalciparum and Toxoplasma gondii.

Interferon alpha: At least 23 different variants of IFN-α are known. Theindividual proteins have molecular masses between 19-26 kDa and consistof proteins with lengths of 156-166 and 172 amino acids. All IFN-αsubtypes possess a common conserved sequence region between amino acidpositions 115-151 while the amino-terminal ends are variable. Many IFN-αsubtypes differ in their sequences at only one or two positions.Naturally occurring variants also include proteins truncated by 10 aminoacids at the carboxy-terminal end.

There are at least 23 different IFN-α genes. They have a length of 1-2kb and are clustered on human chromosome 9p22. Based upon the structurestwo types of IFN-alpha genes, designated class I and II, aredistinguished. They encode proteins of 156-166 and 172 amino acids,respectively.

IFN-α is assayed by a cytopathic effect reduction test employing humanand bovine cell lines. Minute amounts of IFN-α can be assayed also bydetection of the Mx protein specifically induced by this interferon. Asandwich ELISA employing bi-specific monoclonal antibodies for rapiddetection is also available.

Interferon gamma: IFN-γ is a dimeric protein with subunits of 146 aminoacids. The protein is glycosylated at two sites, and the pI is 8.3-8.5.IFN-γ is synthesized as a precursor protein of 166 amino acids includinga secretory signal sequence of 23 amino acids. Two molecular forms ofthe biologically active protein of 20 and 25 kDa have been described.Both of them are glycosylated at position 25. The 25 kDa form is alsoglycosylated at position 97. The observed differences of natural IFN-γwith respect to molecular mass and charge are due to variableglycosylation patterns. 40-60 kDa forms observed under non-denaturingconditions are dimers and tetramers of IFN-γ. The human gene has alength of approximately 6 kb. It contains four exons and maps tochromosome 12q24.1.

IFN-γ can be detected by sensitive immunoassays, such as an ELSA testthat allows detection of individual cells producing IFN-γ. Minuteamounts of IFN-γ can be detected indirectly by measuring IFN-inducedproteins such as Mx protein. The induction of the synthesis of IP-10 hasbeen used also to measure IFN-gamma concentrations. In addition,bioassays can be used to detect IFN-γ, such as an assay that employsinduction of indoleamine 2,3-dioxygenase activity in 2D9 cells.

Interferon Inducible Protein 10: A cytokine that is 98 amino acids inlength that has homology to platelet factor-4, and is a chemokine. Thehuman IP-10 genes contains four exons and maps to chromosome 4q12-21.

Interleukin-6: IL-6 is a cytokine that is 185 amino acids in length.This polypeptide is glycosylated at positions 73 and 172, and issynthesized as a precursor protein of 212 amino acids. Monocytes expressat least five different molecular forms of IL-6 with molecular masses of21.5-28 kDa. They mainly differ by post-translational alterations suchas glycosylation and phosphorylation. IL-6 isolated from various celltypes shows some microheterogeneity in its N-terminus.

The human IL-6 gene has a length of approximately 5 kb and contains fiveexons. It maps to human chromosome 7p21-p14 between the markers D7S135and D7S370. The murine gene maps to chromosome 5. Human IL6 isbiologically active in monkeys, rats, and mice.

IL-6 has a myriad of activities and has been demonstrated to influenceantigen-specific immune responses and inflammatory reactions. It is oneof the major physiological mediators of an acute immune response.

Interleukin-10: IL-10 is a homodimeric protein with subunits having alength of 160 amino acids that is a cytokine. Human IL-10 shows 73percent amino acid homology with murine IL-10. The human IL-10 genecontains four exons.

IL-10 inhibits the synthesis of a number of cytokines such as IL-2 andIFN-γ in Th1 subpopulations of T-cells but not of Th2. IL-10 can bedetected with an ELISA assay. In addition, the murine mast cell line D36can be used to bioassay human IL-10. The intracellular factor can bedetected also by flow cytometry.

Isolated: An “isolated” biological component (such as a nucleic acid,peptide or protein) has been substantially separated, produced apartfrom, or purified away from other biological components in the cell ofthe organism in which the component naturally occurs, i.e., otherchromosomal and extrachromosomal DNA and RNA, and proteins. Nucleicacids, peptides and proteins which have been “isolated” thus includenucleic acids and proteins purified by standard purification methods.The term also embraces nucleic acids, peptides and proteins prepared byrecombinant expression in a host cell as well as chemically synthesizednucleic acids.

K Type Oligodeoxynucleotide (K ODN): An oligodeoxynucleotide includingan unmethylated CpG motif that has a sequence represented by theformula:5′N₁N₂N₃Q-CpG-WN₄N₅N₆3′

wherein the central CpG motif is unmethylated, Q is T, G or A, W is A orT, and N₁, N₂, N₃, N₄, N₅, and N₆ are any nucleotides. In oneembodiment, Q is a T. Additional detailed description of K ODN sequencesand their activities can be found in the section entitled “Multiple Dand K Type Oligodeoxynucleotides (ODN) that Include a CpG Motif.”Generally K ODNs can stimulate a humoral response. For example, K ODNsstimulate the production of IgM.

Leukocyte: Cells in the blood, also termed “white cells,” that areinvolved in defending the body against infective organisms and foreignsubstances. Leukocytes are produced in the bone marrow. There are 5 maintypes of white blood cell, subdivided between 2 main groups:polymorphonuclear leukocytes (neutrophils, eosinophils, basophils) andmononuclear leukocytes (monocytes and lymphocytes). When an infection ispresent, the production of leukocytes increases.

Mammal: This term includes both human and non-human mammals. Similarly,the term “subject” includes both human and veterinary subjects.

Maturation: The process in which an immature cell, such as dendriticcell, changes in form or function to become a functional mature cell,such as an APC.

Neoplasm: An abnormal cellular proliferation, which includes benign andmalignant tumors, as well as other proliferative disorders.

Nucleic acid: A deoxyribonucleotide or ribonucleotide polymer in eithersingle or double stranded form, and unless otherwise limited,encompasses known analogues of natural nucleotides that hybridize tonucleic acids in a manner similar to naturally occurring nucleotides.

Oligonucleotide or “oligo”: Multiple nucleotides (i.e. moleculescomprising a sugar (e.g. ribose or deoxyribose) linked to a phosphategroup and to an exchangeable organic base, which is either a substitutedpyrimidine (Py) (e.g. cytosine (C), thymine (T) or uracil (U)) or asubstituted purine (Pu) (e.g. adenine (A) or guanine (G)). The term“oligonucleotide” as used herein refers to both oligoribonucleotides(ORNs) and oligodeoxynucleotides (ODNs). The term “oligonucleotide” alsoincludes oligonucleosides (i.e. an oligonucleotide minus the phosphate)and any other organic base polymer. Oligonucleotides can be obtainedfrom existing nucleic acid sources (e.g. genomic or cDNA), but arepreferably synthetic (e.g. produced by oligonucleotide synthesis).

A “stabilized oligonucleotide” is an oligonucleotide that is relativelyresistant to in vivo degradation (for example via an exo- orendo-nuclease). In one embodiment, a stabilized oligonucleotide has amodified phosphate backbone. One specific, non-limiting example of astabilized oligonucleotide has a phosphorothioate modified phosphatebackbone (wherein at least one of the phosphate oxygens is replaced bysulfur). Other stabilized oligonucleotides include: nonionic DNAanalogs, such as alkyl- and aryl-phosphonates (in which the chargedphosphonate oxygen is replaced by an alkyl or aryl group),phosphodiester and alkylphosphotriesters, in which the charged oxygenmoiety is alkylated. Oligonucleotides which contain a diol, such astetraethyleneglycol or hexaethyleneglycol, at either or both terminihave also been shown to be substantially resistant to nucleasedegradation.

An “immunostimulatory oligodeoxynucleotide,” “immunostimulatory CpGcontaining oligodeoxynucleotide,” “CpG ODN,” refers to anoligodeoxynucleotide, which contains a cytosine, guanine dinucleotidesequence and (e.g. has a mitogenic effect or induces cytokineproduction) vertebrate immune cells. In one embodiment, animmunostimulatory CpG ODN stimulates a parameter of an immune responsein a subject. The cytosine, guanine is unmethylated.

An “oligonucleotide delivery complex” is an oligonucleotide associatedwith (e.g. ionically or covalently bound to; or encapsulated within) atargeting agent (e.g. a molecule that results in a higher affinitybinding to a target cell (e.g. B-cell or natural killer (NK) cell)surface and/or increased cellular uptake by target cells). Examples ofoligonucleotide delivery complexes include oligonucleotides associatedwith: a sterol (e.g. cholesterol), a lipid (e.g. cationic lipid,virosome or liposome), or a target cell specific binding agent (e.g. aligand recognized by a target cell specific receptor). Preferredcomplexes must be sufficiently stable in vivo to prevent significantuncoupling prior to internalization by the target cell. However, thecomplex should be cleavable or otherwise accessible under appropriateconditions within the cell so that the oligonucleotide is functional.(Gursel, J. Immunol. 167: 3324, 2001)

Pharmaceutical agent or drug: A chemical compound or composition capableof inducing a desired therapeutic or prophylactic effect when properlyadministered to a subject. Pharmaceutical agents include, but are notlimited to, chemotherapeutic agents and anti-infective agents.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers useful in this invention are conventional. Remington'sPharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton,Pa., 15th Edition (1975), describes compositions and formulationssuitable for pharmaceutical delivery of the fusion proteins hereindisclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

Preventing or treating a disease: “Preventing” a disease refers toinhibiting the full development of a disease, for example in a personwho is known to have a predisposition to a disease such as an autoimmunedisorder. An example of a person with a known predisposition is someonewith a history of diabetes in the family, or who has been exposed tofactors that predispose the subject to a condition, such as lupus orrheumatoid arthritis. “Treatment” refers to a therapeutic interventionthat ameliorates a sign or symptom of a disease or pathologicalcondition after it has begun to develop.

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified peptidepreparation is one in which the peptide or protein is more enriched thanthe peptide or protein is in its natural environment within a cell.Preferably, in a purified preparation, the protein or peptide representsat least 50% of the total peptide or protein content of the preparation.Similarly, in a purified preparation of oligodeoxynucleotides, theoligodeoxynucleotide represents at least 50% of the total nucleic acidcontent of the preparation.

Self-complementary nucleic acid sequence: A nucleic acid sequence thatcan form Watson-Crick base pairs. The four bases characteristic ofdeoxyribonucleic unit of DNA are the purines (adenine and guanine) andthe pyrimidines (cytosine and thymine). Adenine pairs with thymine viatwo hydrogen bonds, while guanine pairs with cytosine via three hydrogenbonds. If a nucleic acid sequence includes two or more bases in sequencethat can form hydrogen bonds with two or more other bases in the samenucleic acid sequence, then the nucleic acid includes aself-complementary sequence. In several embodiments, aself-complementary nucleic acid sequence includes 3, 4, 5, 6 or morebases that could form hydrogen bonds with 3, 4, 5, 6 or more bases,respectively, of the same nucleic acid sequence.

Therapeutically effective dose: A dose sufficient to preventadvancement, or to cause regression of a disease, or which is capable ofrelieving symptoms caused by a disease, such as pain or swelling.

Vaccine: A preparation of attenuated microorganisms (including but notlimited to bacteria and viruses), living microorganisms, antigen, orkilled microorganisms, administered for the prevention, amelioration ortreatment of infectious disease.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. It is further tobe understood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. The term “comprises” means “includes.”All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting.

Multiple D and K Type Oligodeoxynucleotides (ODNs) that Include a CpGMotif

Compositions are disclosed herein that include multipleoligodeoxynucleotides with a CpG motif, wherein the sequences of theoligodeoxynucleotides are different. The multiple oligodeoxynucleotidesare either K type CpG ODNs or D type ODNs. These compositions are of usein inducing an immune response in a subject. The compositions include atleast two ODNs including a CpG motif. Thus, for example, thecompositions can include two, three, four, or five ODNs including a CpGmotif. Each of the oligodeoxynucleotides has a different sequence.

I. K Type ODNs

An oligodeoxynucleotide (ODN) composition is disclosed herein thatincludes at least two oligodeoxynucleotides of at least about 10nucleotides in length, wherein the oligodeoxynucleotides comprise anunmethylated CpG motif. In one embodiment, at least one of theoligodeoxynucleotides includes multiple CpG motifs. In anotherembodiment, each of the oligodeoxynucleotide includes a single CpGmotif. The sequence of each of the oligodeoxynucleotides differs fromthe sequence of the other oligodeoxynucleotides.

In one embodiment, the ODNs are K type ODNs. K ODNs which exhibit thegreatest immunostimulatory activity share specific characteristics.These characteristics differ from those of the Formula II or D ODN (seebelow). In addition, K ODN have specific effects on the cells of theimmune system, which differ from the effects of D ODN. For example, KODN stimulate production of IgM.

The K ODNs are least about 10 nucleotides in length and include asequence represented by either Formula I:5′N₁N₂N₃Q-CpG-WN₄N₅N₆3′wherein the central CpG motif is unmethylated, Q is T, G or A, W is A orT, and N₁, N₂, N₃, N₄, N₅, and N₆ are any nucleotides.

These Formula I or K ODN can stimulate B cell proliferation and thesecretion of IgM and IL-6, and processes involved in the body's humoralimmunity, such as the production of antibodies against foreign antigens.In one embodiment, the K ODNs induce a humoral immune response.

In one embodiment, K type oligodeoxynucleotides include a sequencerepresented by the formula5′N₁N₂N₃T-CpG-WN₄N₅N₆3′.In another embodiment, K type oligodeoxynucleotides include a phosphatebackbone modification. In one specific, non-limiting example, thephosphate backbone modification is a phosphorothioate backbonemodification (i.e., one of the non-bridging oxygens is replaced withsulfur, as set forth in International Patent Application WO 95/26204,herein incorporated by reference). In one embodiment, K ODNs have aphosphorothioate backbone, and at least one unmethylated CpGdinucleotide. Eliminating the CpG dinucleotide motif from the K ODNsignificantly reduces immune activation. Incorporating multiple CpGs ina single K ODN can increase immune stimulation. In one embodiment, the KODN are at least 12 bases long. In addition, K ODN containing CpG motifsat the 5′ end are the most stimulatory, although at least one baseupstream of the CpG is required. More particularly, the most active KODNs contain a thymidine immediately 5′ from the CpG dinucleotide, and aTpT or a TpA in a position 3′ from the CpG motif. Modifications whichare greater than 2 base pairs from the CpG dinucleotide motif appear tohave little effect on K ODN activity.

K-type CpG oligodeoxynucleotides can include modified nucleotides. Anysuitable modification can be used to render the ODN resistant to in vivodegradation resulting from, e.g., exo or endonuclease digestion. In oneembodiment, the modification includes a phosphorothioate modification.The phosphorothioate modifications can occur at either termini, e.g.,the last two or three 5′ and/or 3′ nucleotides can be linked withphosphorothioate bonds. The ODN also can be modified to contain asecondary structure (e.g., stem loop structure) such that it isresistant to degradation. Another modification that renders the ODN lesssusceptible to degradation is the inclusion of nontraditional bases suchas inosine and quesine, as well as acetyl-, thio- and similarly modifiedforms of adenine, cytidine, guanine, thymine, and uridine. Othermodified nucleotides include nonionic DNA analogs, such as alkyl or arylphosphonates (i.e., the charged phosphonate oxygen is replaced with analkyl or aryl group, as set forth in U.S. Pat. No. 4,469,863),phosphodiesters and alkylphosphotriesters (i.e., the charged oxygenmoiety is alkylated, as set forth in U.S. Pat. No. 5,023,243 andEuropean Patent No. 0 092 574). ODNs containing a diol, such astetraethyleneglycol or hexaethyleneglycol, at either or both termini,have also been shown to be more resistant to degradation.

In one embodiment, a composition is provided that includes multipledifferent K type ODN (for example two, three, four or more different Ktype ODN). Thus, disclosed herein is an oligodeoxynucleotide compositionincluding at least two different oligodeoxynucleotide of at least about10 nucleotides in length, wherein the oligodeoxynucleotides include anunmethylated CpG motif that can be used to induce an immune response ina subject. Thus, the at least two ODNs in the composition include asequence represented by the formula 5′ N₁N₂N₃T-CpG-WN₄N₅N₆ 3′, wherein Wis A or T, and N₁, N₂, N₃, N₄, N₅, and N₆ are any nucleotide. In oneembodiment, at least one of the oligodeoxynucleotides includes multipleCpG motifs. In another embodiment, the oligodeoxynucleotides eachinclude only a single CpG motif. In these embodiments, the two or moreoligodeoxynucleotides differ in their nucleic acid sequence.

One specific non-limiting example is a composition that includes anoligodeoxynucleotide including a sequence as set forth as SEQ ID NO: 1,an oligodeoxynucleotide including a sequence as set forth as SEQ ID NO:8, and an oligodeoxynucleotide including a sequence as set forth as SEQID NO: 20. Another specific non-limiting example is a composition thatincludes an oligodeoxynucleotide including a sequence as set forth asSEQ ID NO: 1, an oligodeoxynucleotide including a sequence as set forthas SEQ ID NO: 20, and an oligodeoxynucleotide including a sequence asset forth as SEQ ID NO: 108. Yet another specific non-limiting exampleis a composition that includes an oligodeoxynucleotide including asequence as set forth as SEQ ID NO: 1, an oligodeoxynucleotide includinga sequence as set forth as SEQ ID NO: 7, and an oligodeoxynucleotideincluding a sequence as set forth as SEQ ID NO: 108. A further specificnon-limiting example is a composition, that includes anoligodeoxynucleotide including a sequence as set forth as SEQ ID NO: 7,an oligodeoxynucleotide including a sequence as set forth as SEQ ID NO:20, and an oligodeoxynucleotide including a sequence as set forth as SEQID NO: 98. Another specific, non-limiting example is a composition thatincludes an oligodeoxynucleotide including a sequence as set forth asSEQ ID NO: 20, an oligodeoxynucleotide including a sequence as set forthas SEQ ID NO: 98, and including an oligodeoxynucleotide having asequence as set forth as SEQ ID NO: 108. Yet another specific,non-limiting example is a composition that includes anoligodeoxynucleotide including a sequence as set forth as SEQ ID NO: 1,an oligodeoxynucleotide including a sequence as set forth as SEQ ID NO:7, and an oligodeoxynucleotide including a sequence as set forth as SEQID NO: 20.

II. D Type ODNs

D type ODNs differ both in structure and activity from K type ODNs. Theunique activities of D type ODNs are disclosed below (see section C).For example, as disclosed herein, D oligodeoxynucleotides stimulate therelease of cytokines from cells of the immune system. In specific,non-limiting examples D type oligodeoxynucleotides stimulate the releaseor production of IFN-α by monocytes and/or plasmacitoid dendritic cellsand the release or production of IFN-γ by NK cells. The stimulation ofNK cells by D oligodeoxynucleotides can be either direct or indirect.

With regard to structure, in one embodiment, a CpG motif in a D typeoligodeoxynucleotides has been described by Formula II:5′RY-CpG-RY3′wherein the central CpG motif is unmethylated, R is A or G (a purine),and Y is C or T (a pyrimidine). D-type oligodeoxynucleotides include anunmethylated CpG dinucleotide. Inversion, replacement or methylation ofthe CpG reduces or abrogates the activity of the D ODN.

In one embodiment, a D type ODN is at least about 16 nucleotides inlength and includes a sequence represented by Formula III:5′X₁X₂X₃Pu₁Py₂CpGPu₃Py₄X₄X₅X₆(W)_(M)(G)_(N)-3′wherein the central CpG motif is unmethylated, Pu is a purinenucleotide, Py is a pyrimidine nucleotide, X and W are any nucleotide, Mis any integer from 0 to 10, and N is any integer from 4 to 10.

The region Pu₁ Py₂ CpG Pu₃ Py₄ is termed the CpG motif. The regionX₁X₂X₃ is termed the 5′ flanking region, and the region X₄X₅X₆ is termedthe 3′ flanking region. If nucleotides are included 5′ of X₁X₂X₃ in theD ODN these nucleotides are termed the 5′ far flanking region.Nucleotides 3′ of X₄X₅X₆ in the D ODN are termed the 3′ far flankingregion.

In one specific non-limiting example, Py₂ is a cytosine. In anotherspecific, non-limiting example, Pu₃ is a guanidine. In yet anotherspecific, non limiting example, Py₂ is a thymidine and Pu₃ is anadenine. In a further specific, non-limiting example, Pu₁ is an adenineand Py₂ is a tyrosine. In another specific, non-limiting example, Pu₃ isan adenine and Py₄ is a tyrosine.

In one specific non-limiting example, N is from about 4 to about 8. Inanother specific, non-limiting example, N is about 6.

D-type CpG oligodeoxynucleotides can include modified nucleotides.Without being bound by theory, modified nucleotides can be included toincrease the stability of a D-type oligodeoxynucleotide. Without beingbound by theory, because phosphorothioate-modified nucleotides conferresistance to exonuclease digestion, the D ODN are “stabilized” byincorporating phosphorothioate-modified nucleotides. In one embodiment,the CpG dinucleotide motif and its immediate flanking regions includephosphodiester rather than phosphorothioate nucleotides. In one specificnon-limiting example, the sequence Pu₁ Py₂ CpG Pu₃ Py₄ includesphosphodiester bases. In another specific, non-limiting example, all ofthe bases in the sequence Pu₁ Py₂ CpG Pu₃ Py₄ are phosphodiester bases.In yet another specific, non-limiting example, X₁X₂X₃ andX₄X₅X₆(W)_(M)(G)_(N) include phosphodiester bases. In yet anotherspecific, non-limiting example, X₁X₂X₃ Pu₁ Py₂ CpG Pu₃ Py₄X₄X₅X₆(W)_(M)(G)_(N) include phosphodiester bases. In furthernon-limiting examples the sequence X₁X₂X₃ includes at most one or atmost two phosphothioate bases and/or the sequence X₄X₅X₆ includes atmost one or at most two phosphothioate bases. In additional non-limitingexamples, X₄X₅X₆(W)_(M)(G)_(N) includes at least 1, at least 2, at least3, at least 4, or at least 5 phosphothioate bases. Thus, a D typeoligodeoxynucleotide can be a phosphorothioate/phosphodiester chimera.

As disclosed herein, any suitable modification can be used in thepresent invention to render the ODNs resistant to degradation in vivo(e.g., via an exo- or endo-nuclease). In one specific, non-limitingexample, a modification that renders the oligodeoxynucleotide lesssusceptible to degradation is the inclusion of nontraditional bases suchas inosine and quesine, as well as acetyl-, thio- and similarly modifiedforms of adenine, cytidine, guanine, thymine, and uridine. Othermodified nucleotides include nonionic DNA analogs, such as alkyl or arylphosphonates (i.e., the charged phosphonate oxygen is replaced with analkyl or aryl group, as set forth in U.S. Pat. No. 4,469,863),phosphodiesters and alkylphosphotriesters (i.e., the charged oxygenmoiety is alkylated, as set forth in U.S. Pat. No. 5,023,243 andEuropean Patent No. 0 092 574). Oligonucleotides containing a diol, suchas tetraethyleneglycol or hexaethyleneglycol, at either or both termini,have also been shown to be more resistant to degradation. The D typeoligodeoxynucleotides can also be modified to contain a secondarystructure (e.g., stem loop structure). Without being bound by theory, itis believed that incorporation of a stem loop structure renders andoligodeoxynucleotide more effective.

In a further embodiment, Pu₁ Py₂ and Pu₃ Py₄ are self-complementary. Inanother embodiment, X₁X₂X₃ and X₄X₅X₆ are self-complementary. In yetanother embodiment X₁X₂X₃ Pu₁ Py₂ and Pu₃ Py₄ X₄X₅X₆ areself-complementary.

In one embodiment, the D type oligodeoxynucleotides disclosed herein areat least about 16 nucleotides in length. In a second embodiment, a Dtype oligodeoxynucleotide is at least about 18 nucleotides in length. Inanother embodiment, a D type oligodeoxynucleotide is from about 16nucleotides in length to about 100 nucleotides in length. In yet anotherembodiment, a D type oligodeoxynucleotide is from about 16 nucleotidesin length to about 50 nucleotides in length. In a further embodiment, aD type oligodeoxynucleotide is from about 18 nucleotides in length toabout 30 nucleotides in length.

In another embodiment, the oligodeoxynucleotide is at least 18nucleotides in length, and at least two G's are included at the 5′ endof the molecule, such that the oligodeoxynucleotide includes a sequencerepresented by Formula IV:5′GGX₁X₂X₃Pu₁Py₂CpGPu₃Py₄X₄X₅X₆(W)_(M)(G)_(N)-3′.

The D type oligodeoxynucleotide can include additional G's at the 5′ endof the oligodeoxynucleotide. In one specific example, about 1 or about 2G's are included at the 5′ end of an oligodeoxynucleotide including asequence as set forth as Formula IV.

Specific, non-limiting examples of D type ODNs can be found in U.S.patent application Ser. No. 10/068,160, which is herein incorporated byreference. Specific, non-limiting examples of D and K type ODN, asdisclosed herein, can be found in Verthelyi et al., J. Immunol.166:2372-2377, 2001, which is herein incorporated by reference.

Thus disclosed herein is an oligodeoxynucleotide composition includingat least two oligodeoxynucleotide of at least about 10 nucleotides inlength, wherein the oligodeoxynucleotides comprise an unmethylated CpGmotif and at least one of the oligodeoxynucleotides has a sequencerepresented by the formula 5′ RY-CpG-RY 3′, wherein R is A or G and Y isC or T. In addition, the oligodeoxynucleotides differ in their nucleicacid sequence. Also disclosed are oligodeoxynucleotide compositionsincluding at least three, or at least four, different D type ODN.

In one embodiment, at least one of the oligodeoxynucleotides is at leastabout 16 nucleotides in length and includes a sequence represented bythe following formula:5′X₁X₂X₃Pu₁Py₂CpGPu₃Py₄X₄X₅X₆(W)_(M)(G)_(N)-3′wherein the central CpG motif is unmethylated, Pu is a purinenucleotide, Py is a pyrimidine nucleotide, X and W are any nucleotide, Mis any integer from 0 to 10, and N is any integer from 4 to 10. Thesecompositions are of use in stimulating an immune response.

In one specific, non-limiting example is a composition that includes anoligodeoxynucleotide including a sequence as set forth as SEQ ID NO: 32,an oligodeoxynucleotide including a sequence as set forth as SEQ ID NO:106, and an oligodeoxynucleotide including a sequence as set forth asSEQ ID NO: 40. Another specific, non-limiting example is a compositionthat includes an oligodeoxynucleotide including a sequence as set forthas SEQ ID NO: 32, an oligodeoxynucleotide including a sequence as setforth as SEQ ID NO: 106, and an oligodeoxynucleotide including asequence as set forth as SEQ ID NO: 102. Yet another specific,non-limiting example is a composition that includes anoligodeoxynucleotide including a sequence as set forth as SEQ ID NO: 32,an oligodeoxynucleotide including a sequence as set forth as SEQ ID NO:42, and an oligodeoxynucleotide including a sequence as set forth as SEQID NO: 40.

The oligodeoxynucleotides (both K and D type) disclosed herein can besynthesized de novo using any of a number of procedures well known inthe art. For example, the oligodeoxynucleotides can be synthesized asset forth in U.S. Pat. No. 6,194,388, which is herein incorporated byreference in its entirety. A oligodeoxynucleotide including a CpG motifcan be synthesized using, for example, the B-cyanoethyl phosphoramiditemethod or nucleoside H-phosphonate method. These chemistries can beperformed by a variety of automated oligonucleotide synthesizersavailable in the market. Alternatively, oligodeoxynucleotides can beprepared from existing nucleic acid sequences (e.g. genomic or cDNA)using known techniques, such as employing restriction enzymes,exonucleases or endonucleases, although this method is less efficientthan direct synthesis.

Delivery Complexes and Pharmaceutical Compositions

In one embodiment, multiple K type or D type oligodeoxynucleotides (ODN)including a CpG motif are included in a delivery complex. The deliverycomplex can include the multiple different ODN (e.g. at least twodifferent ODNs, at least three different ODNs, or at least fourdifferent ODNs) and a targeting agent. A targeting agent is any agentthat can be used to increase the delivery of an ODN to a cell. The cellcan be any cell, including, but not limited to an antigen presentingcell, a dendritic cell, a B cell or a T cell. Any suitable targetingagent can be used.

For example, one or more of the oligodeoxynucleotides can be associatedwith (e.g., ionically or covalently bound to, or encapsulated within) atargeting agent. A variety of coupling or cross-linking agents can beused to form the delivery complex, such as protein A, carbodiamide, andN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP). Examples ofoligodeoxynucleotide delivery complexes include an oligodeoxynucleotideassociated with a sterol (e.g., cholesterol), a lipid (e.g., a cationiclipid, anionic lipid, virosome or liposome), and a target cell specificbinding agent (e.g., a ligand recognized by target cell specificreceptor). Without being bound by theory, the complex is sufficientlystable in vivo to prevent significant uncoupling prior to delivery tothe target cell. In one embodiment, the delivery complex is cleavablesuch that the oligodeoxynucleotide is released in a functional form atthe target cells.

In one embodiment, a pharmacological composition is provided thatincludes at least two oligodeoxynucleotides and a pharmacologicallyacceptable carrier. Pharmacologically acceptable carriers (e.g.,physiologically or pharmaceutically acceptable carriers) are well knownin the art. A suitable pharmacological composition can be formulated tofacilitate the use of a K type ODN or D type ODN in vivo and/or ex vivo.Such a composition can be suitable for delivery of the active ingredientto any suitable host, such as a patient or other subject for medicalapplication, and can be manufactured in a manner that is itself known,e.g., by means of conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping orlyophilizing processes.

Pharmacological compositions for use can be formulated in a conventionalmanner using one or more pharmacologically (e.g., physiologically orpharmaceutically) acceptable carriers including excipients, as well asoptional auxiliaries that facilitate processing of the active compoundsinto preparations which can be used pharmaceutically. Proper formulationis dependent upon the route of administration chosen, and whether usewill be an in vivo or an ex vivo use. For use in vivo, administrationcan be either systemic or local. In addition, one of skill in the artcan readily select a suitable route of administration, including, butnot limited to intravenous, intramuscular, intraperitioneal,transmucosal, subcutaneous, transdermal, transnasal, and oraladministration.

Thus, for injection, at least two ODNs including a CpG motif can beformulated in aqueous solutions, preferably in physiologicallycompatible buffers. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art. For oral administration,the active ingredient can be combined with carriers suitable forinclusion into tablets, pills, dragees, capsules, liquids, gels, syrups,slurries, suspensions and the like. For administration by inhalation,the active ingredient is conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebuliser, withthe use of a suitable propellant. The active ingredient can beformulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Similarly, D type or K typeoligodeoxynucleotides can be formulated for intratracheal administrationor for inhalation. Such compositions can take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and can containformulatory agents such as suspending, stabilizing and/or dispersingagents. Other pharmacological excipients are known in the art.

Compositions that Include Multiple K Type or Multiple D Type ODN andTheir Use in Inducing an Immune Response

Compositions including multiple different ODN can be administered to asubject to induce an immune response. The administration of the multipledifferent oligodeoxynucleotides can be by any suitable method. Forexample, the ODNs can be administered in vivo or ex vivo. The subjectcan be any mammal, particularly a primate, such as a human.

In one embodiment, the compositions are of use in inducing an immuneresponse in a subject. A substantial “induction” or an “increase” in animmune response can be, for example, at least an about 50%, 75%, 90%,100%, 200%, 300%, or 500% increase in a single parameter of an immuneresponse. In one embodiment, administration of a composition includingmultiple different ODNs that include a CpG motif results in asubstantial increase in a single parameter of an immune response ascompared to administration of one of the ODNs of the composition. Inanother embodiment, administration of a composition that includesmultiple different ODNs including a CpG motif results in a substantialincrease in two or more parameters of an immune response as compared toadministration of one of the ODNs of the composition. In a furtherembodiment, administration of the composition including multipledifferent ODNs with a CpG motif increases more parameters of the immuneresponse than the administration of only one of the ODNs of thecomposition. Thus, administration of multiple different ODNs with a CpGmotif broadens the immune response in a subject as compared toadministration of a single ODN.

In another embodiment, the compositions disclosed herein are of use ininducing an immune response a large percentage of the individuals in apopulation. Thus, in one embodiment, upon administration of the multipleoligodeoxynucleotides to a population of subjects, at least about 80%,at least about 90%, at least about 95%, at least about 98%, or at leastabout 99%, of the individuals in the population produce an immuneresponse. In one embodiment, following administration of the multipledifferent oligodeoxynucleotides to a population of subjects, at leastabout 80% of the subjects produce an immune response. In one embodiment,the immune response includes substantial stimulation of a singleparameter of the immune response as compared to administration of onlyone of the oligodeoxynucleotides to the population of subjects. Inanother embodiment, the immune response includes substantial stimulationof two or more parameters of the immune response as compared toadministration of only one of the oligodeoxynucleotides to thepopulation of subjects. In another embodiment, upon administration ofthe multiple different oligodeoxynucleotides to a population ofsubjects, at least two parameters of the immune response are stimulatedin the subjects, wherein administration of only one of theoligodeoxynucleotides of the composition to the population of subjectsstimulates only a single parameter of the immune response.

In order to induce an immune response, the multiple different ODNsincluding a CpG motif can be administered either alone or in conjunctionwith another molecule. Co-administration includes administering themolecule and the multiple different ODNs including the CpG motif at thesame time, or sequentially, for example, substantiallycontemporaneously, with the other molecule. The other molecule can beany other agent, such as a protein, an antigenic epitope, a hydrocarbon,lipid, mitogen, an anti-infectious agent (such as antiviral, antifungal,or anti-bacterial agent) a anti-neoplastic agent, or a vaccine (such asa live, attenuated, or heat-killed vaccine).

In one embodiment, at least two different ODNs including a CpG motif areadministered to a subject, such as a subject that has an autoimmunedisease. Specific, non-limiting examples of autoimmune diseases include,but are not limited to diabetes, rheumatoid arthritis, lupuserythematosus, and multiple sclerosis.

Also disclosed herein are methods of use to treat, prevent, orameliorate an allergic reaction in a subject. An allergy refers to anacquired hypersensitivity to a substance (i.e., an allergen). Allergicconditions include eczema, allergic rhinitis or coryza, hay fever,bronchial asthma, urticaria (hives), food allergies, and other atopicconditions. The list of allergens is extensive and includes pollens,insect venoms, animal dander, dust, fungal spores, and drugs (e.g.,penicillin). Examples of natural, animal, and plant allergens can befound in International Patent Application WO 98/18810. In one embodimentat least two different oligodeoxynucleotides including a CpG motif areadministered to a subject to treat an allergic condition such asallergic asthma. In another embodiment, the ODNs are administered incombination with any suitable anti-allergenic agent. Suitableanti-allergenic agents include those substances given in treatment ofthe various allergic conditions described above, examples of which canbe found in the Physicians' Desk Reference (1998).

In another embodiment, at least two different ODNs including a CpG motifare administered to a subject that has a neoplasm. The ODNs areadministered either alone or in combination with any suitableanti-neoplastic agent, such as a chemotherapeutic agent or radiation.Suitable neoplasms include, but are not limited to, solid tumors such ascancers of the brain, lung (e.g., small cell and non-small cell), ovary,breast, prostate, and colon, as well as carcinomas and sarcomas. Withoutbeing bound by theory, it is believed that the ODNs increase the immuneresponse to the neoplasm, and thus are involved in the reduction oftumor burden.

In a further embodiment, a method is provided to enhance the efficacy ofany suitable vaccine. Suitable vaccines include those directed againstLeishmania, Hepatitis A, B, and C, examples of which can be found in thePhysicians' Desk Reference (1998), and DNA vaccines directed against,for example, HIV and malaria. (See generally Klinman et al., 17 Vaccine17:19 (1999); McCluskie and Davis, J. Immun. 161:4463 (1998)).

Multiple different ODNs including a CpG motif can be used to treat,prevent, or ameliorate any condition associated with an infectiousagent. The multiple different ODNs can be administered to a subjectinfected with the infectious agent alone or in combination with anysuitable anti-infectious agent, such as an antiviral, anti-fungal oranti-bacterial agent (see Physicians' Desk Reference, 1998). Specific,non-limiting examples of infectious agents, and conditions associatedwith infectious agents are tularemia, francisella, schistosomiasis,tuberculosis, malaria, and leishmaniasis. Examples of infectious agentssuch as viruses, bacteria, fungi, and other organisms (e.g., protists)can be found in International Patent Application WO 98/18810.

Compositions including multiple ODNs with CpG motifs can be used to animmune response in combination with any suitable antisense therapy.Suitable antisense agents are those that bind either with DNA or RNA andblock their function by inhibiting expression of the sequence to whichthe antisense agents are bound. See generally Lonnberg et al., 28 Ann.Med. 511 (1996); Alama et al., 36 Pharmacol. Res. 171 (1997); Scanlon etal., 9 FASEB J. 1288 (1995), amongst others.

The methods disclosed herein for inducing an immune response can be usedto treat, prevent, or ameliorate the symptoms resulting from exposure toa bio-warfare agent. Suitable bio-warfare agents include those naturallyoccurring biological agents that have been specifically modified in thelaboratory. Often, modification of these agents has altered them suchthat there is no known treatment. Examples include Ebola, Anthrax, andListeria. In the course of ameliorating the symptoms after exposure,administration of multiple ODNs including a CpG motif may not cure thepatient, but rather can extend the patient's life sufficiently such thatsome other treatment can then be applied.

In one embodiment the different ODNs are K type ODN and a parameter ofthe humoral immune response is substantially increased in the subject.Parameters of the humoral immune response include, but are not limitedto, IgM production, Il-6 production, and/or proliferation. In oneembodiment, the multiple different ODN are K type ODN and the immuneresponse is a humoral immune response. Thus, in one embodiment, theimmune response comprises proliferation of peripheral blood mononuclearcells, IgM production, IL-6 production, or a combination thereof. Thus,in one specific, non-limiting example, K type ODN can be selected suchthat administration of the combination of K type ODN causes increasedproduction of IL-6 in a at least about 80%, at least about 90%, at leastabout 95%, at least about 98%, or at least about 99%, of the individualsin the population produces an immune response.

In another embodiment, multiple different D type ODNs are used toproduce a substantial immune response in a subject. Administration of aD type oligodeoxynucleotide activates monocytes and/or natural killercells, and induces the maturation of dendritic cells. Furthermore, a Dtype oligodeoxynucleotide can be used to increase the production ofcytokines (for example IP-10, IFN-α or IFN-γ) by a cell of the immunesystem.

In one embodiment, a method is provided for inducing an immune responsein a subject wherein the method includes contacting a monocyte or adendritic cell precursor in vitro with multiple different D type ODNs toproduce an activated antigen presenting cell. The monocytes or dendriticcell precursors can be contacted with the D type ODNs in the presence ofor in the absence of antigen. The activated antigen presenting cell isthen administered to the subject to induce an immune response.

In another embodiment, a method is provided herein for inducing animmune response in a subject that includes contacting a monocyte or adendritic cell precursor in vitro with a D type ODNs to produce anactivated antigen presenting cell. The monocytes or dendritic cellprecursors can be contacted with the D type ODNs in the presence of orin the absence of antigen. Lymphocytes or natural killer are thencontacted with the activated antigen presenting cells in vitro, or withcytokines secreted by the activated antigen presenting cells in vitro,to produce activated lymphocytes or activated natural killer cells. Theactivated lymphocytes or natural killer cells are then administered tothe subject to induce the immune response. In yet another embodiment, animmunoregulatory response is induced.

This disclosure is illustrated by the following non-limiting Examples.

EXAMPLES Example 1

The following example demonstrates the varied immune response induced invitro in different donors after administration of an ODN including asingle CpG sequence. Induction of an immune response was measured byproduction of the cytokines IL-6 and IFN-γ, and cell proliferation inhuman peripheral blood mononuclear cells (PBMC) isolated from individualdonors.

PBMC were isolated, as described elsewhere (Ballas et al., 85 J. AllergyClin. Immunol. 453 (1990); Ballas et al., 45 1039 (1990); Ballas et al.,150 J. Immunol. 17 (1993)). ODNs were synthesized on a DNA synthesizer(Applied Biosystems Inc., Foster City, Calif.), as described elsewhere(Beacage and Caruthers, “Deoxynucleoside Phosphoramidites—A New Class ofKey Intermediates for Deoxypolynucleotide Synthesis,” 22 TetrahedronLetters 1859 (1981)). In some ODNs, the normal DNA backbonephosphodiesterase linkages were replaced with phosphorothioate linkages,as described elsewhere (Agrawal et al., 94 Proc. Natl. Acad. Sci. USA2620 (1997); Agrawal 14 TIB TECH 376 (1996)). To reduce degradation ofthe ODNs, those that did not have an entire phosphorothioate backbonecontained phosphorothioate linkages at the 5′ and 3′ ends. Cells wereincubated for approximately 72 hours with the various ODNs. IL-6 andIFN-γ levels were determined by ELISA using anti-IL-6 and anti-IFN-γantibodies, as described elsewhere (Maniatis et al., Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York,1989). Cell proliferation was determined by [³H] thymidineincorporation, as described elsewhere (Liang et al., 98 J. Clin. Invest.at 1121). IL-6 levels are set forth in Table 1: Induction of an ImmuneResponse (IL-6); IFN-γ levels are set forth in Table 5: Induction of anImmune Response (IFN-γ); and cell proliferation is set forth in Table 6:Induction of an Immune Response (Cell Proliferation).

TABLE 1 Induction of an Immune Response (IL-6) 3 4 7 8 15 16 17 18 19 2324 25 26 27 IL-6 SEQ ID NO: 1 35 53 19 12 9 2 8 6 33 15 5 40 13 3 SEQ IDNO: 18 2 3 25 65 — — — — — 8 3 3 1 1 SEQ ID NO: 20 50 45 9 43 5 9 20 2020 — 12 57 13 3 SEQ ID NO: 98 18 42 9 41 60 80 25 11 22 17 6 12 2 1 SEQID NO: 105 — — — — — — — — — 13 7 16 3 1

TABLE 2 Induction of an Immune Response (IFN-γ) 3 4 5 9 13 14 15 17IFN-γ (ELISA) SEQ ID NO: 40 154 64 6 2 5 11 15 3 SEQ ID NO: 42 92 56 41928 8 4 4 8 SEQ ID NO: 43 13 3 269 93 15 6 5 8 SEQ ID NO: 100 22 2 16 2 99 9 6 SEQ ID NO: 101 3 2 15 25 0 16 5 5 SEQ ID NO: 102 — — 0 1 60 250 63 SEQ ID NO: 103 — — 4 1 3 2 8 5 SEQ ID NO: 104 — — 4 1 2 1 3 4

TABLE 3 Induction of an Immune Response (Cell Proliferation) 1 2 3 4 2021 22 Proliferation ([³H] T) SEQ ID NO: 1 22 36 33 9 18 15 14 SEQ ID NO:9 5 17 16 11 18 18 13 SEQ ID NO: 10 9 10 20 25 14 23 12 SEQ ID NO: 12 68 10 19 4 7 6 SEQ ID NO: 15 9 47 11 13 14 17 12 SEQ ID NO: 31 2 15 10 166 8 7 SEQ ID NO: 45 3 7 16 15 10 16 10 SEQ ID NO: 50 3 6 10 9 6 6 5 SEQID NO: 54 4 5 10 9 5 5 4

The foregoing data demonstrates induction of an immune response, to anODN that includes various sequences, in human PBMC isolated fromindividual donors. Specifically, these data demonstrate that a singleODN sequence induces a varied immune response in different donors. Forexample, in Table 4, an ODN including SEQ ID NO: 98 induced IL-6 levelsranging from 2 to 80, in Table 5, an ODN including SEQ ID NO: 42 inducedIFN-γ levels ranging from 4 to 419, and in Table 6, an ODN including SEQID NO: 15 induced cell proliferation ranging from 9 to 47.

Example 2

The following example demonstrates in vitro induction of an immuneresponse after administration of an ODN that includes a CpG sequence. Ina population of subjects, only some of the subjects mounted an immuneresponse to each individual ODN. Induction of an immune response wasmeasured by production of the cytokines IL-6 and IFN-γ, and cellproliferation in human PBMC isolated from individual donors.

Human PBMC were isolated, as described in Example 1. ODNs weresynthesized on a DNA synthesizer (Applied Biosystems Inc., Foster City,Calif.), as described in Example 1. In some ODNs, the normal DNAbackbone phosphodiesterase linkages were replaced with phosphorothioatelinkages, as described in Example 1. To reduce degradation of the ODNs,those that did not have an entire phosphorothioate backbone containedphosphorothioate linkages at the 5′ and 3′ ends. Cells were incubatedfor approximately 72 hours with the various ODNs. IL-6 and IFN-γ levelswere determined by ELISA using anti-IL-6 and anti-IFN-γ antibodies, asdescribed in Example 1. Cell proliferation was determined by [³H]thymidine incorporation, as described in Example 1. Results are setforth in Table 4: Induction of an Immune Response to Multiple ODNs.

TABLE 4 Percent Induction of an Immune Response (IL-6) Percent InductionNumber of Donors SEQ ID NO: 1 69% 26 SEQ ID NO: 109 67% 9 SEQ ID NO: 2065% 34 SEQ ID NO: 7 49% 39 SEQ ID NO: 108 47% 38 SEQ ID NO: 98 44% 39SEQ ID NO: 110 30% 10 SEQ ID NO: 111 26% 19 SEQ ID NO: 112  7% 29

TABLE 5 Percent Induction of an Immune Response (IFN-γ) PercentInduction Number of Donors SEQ ID NO: 43 93% 42 SEQ ID NO: 42 91% 23 SEQID NO: 106 87% 23 SEQ ID NO: 32 82% 17 SEQ ID NO: 40 79% 19 SEQ ID NO:103 58% 12 SEQ ID NO: 102 57% 28 SEQ ID NO: 107 10% 31 SEQ ID NO: 68 11%19

TABLE 6 Heterogeneity in Induction of an Immune Response (IL-6) 1 2 3 45 6 7 8 SEQ ID NO: 20 — ++ + + + + ++ ++ SEQ ID NO: 7 + + + — ++ + ++ +SEQ ID NO: 1 ++ ++ + — + + ++ —

TABLE 7 Heterogeneity in Induction of an Immune Response (IFN-γ) 1 2 3 45 6 7 8 SEQ ID NO: 40 ++ ++ — — — + ++ + SEQ ID NO: 32 + ++ + + ++ + — —SEQ ID NO: 102 — — ++ ++ ++ + — ++

The foregoing data demonstrates induction of an immune response to anODN including a CpG motif in human PBMC isolated from individual donors.Specifically, these data demonstrate that a single sequence induces avaried immune response in different donors, as shown, e.g., in Table 4,the percent of donors induced varied from 7% to 69%, as measured by IL-6production, and in Table 5, the percent of donors induced varied from11% to 93%, as measured by IFN-γ production. Further, as demonstrated inTables 6 and 7, there was substantial heterogeneity in induction of animmune response in different donors.

Example 3

The following example demonstrates in vitro induction of an immuneresponse after administration of multiple ODNs, each of which includes adifferent CpG sequence. Induction of an immune response was measured byproduction of the cytokines IL-6 and IFN-γ, and cell proliferation inhuman PBMC isolated from individual donors.

Human PBMC were isolated, as described in Example 1. ODNs weresynthesized on a DNA synthesizer (Applied Biosystems Inc., Foster City,Calif.), as described in Example 1. In some ODNs, the normal DNAbackbone phosphodiesterase linkages were replaced with phosphorothioatelinkages, as described in Example 1. To reduce degradation of the ODNs,those that did not have an entire phosphorothioate backbone containedphosphorothioate linkages at the 5′ and 3′ ends. Cells were incubatedfor approximately 72 hours with the various ODNs. IL-6 and IFN-γ levelswere determined by ELISA using anti-IL-6 and anti-IFN-γ antibodies, asdescribed in Example 1. Cell proliferation was determined by [³H]thymidine incorporation, as described in Example 1. Results are setforth in Table 8: Induction of an Immune Response to Multiple DifferentODNs.

TABLE 8 Induction of an Immune Response to Multiple Different ODNs IL-6IFN-γ Proliferation (ELISA) (ELISA) ([³H] T) Donor 1 SEQ ID NO: 1 12.02.3 13.0 SEQ ID NO: 43 7.0 9.0 1.6 SEQ ID NO: 1 + SEQ ID NO: 43 27 40.019.9 Donor 2 SEQ ID NO: 1 5.0 13.0 37.7 SEQ ID NO: 43 2.0 7.0 1.3 SEQ IDNO: 1 + SEQ ID NO: 43 9.0 33.0 68.6 Donor 3 SEQ ID NO: 1 10.0 8.0 13.1SEQ ID NO: 43 1.0 12.0 1.3 SEQ ID NO: 1 + SEQ ID NO: 43 8.0 42.0 17.5

The foregoing data demonstrates the induction of an immune responseafter administration of multiple different ODNs that include various CpGsequences in human PBMC isolated from individual donors. Specifically,these data demonstrate that multiple ODNs synergistically induce animmune response, as demonstrated by, e.g., an increase of 26.6% asmeasured by cell proliferation, 29.6% as measured by IL-6 levels, and71.7% as measured by IFN-γ levels after administration to Donor 1.Combination of an ODN that includes SEQ ID NO: 1 and an ODN thatincludes SEQ ID NO: 43 produced this enhanced immune response comparedto the combined immune response of each ODN administered separately.

Example 4

The following example demonstrates in vitro induction of an immuneresponse after administration of multiple different ODNs that include aCpG sequence. The induced immune response was measured by increasedIFN-γ in human PBMC isolated from individual donors as compared tounstimulated PBMC from the same donor.

Human PBMC were isolated, as described in Example 1. ODNs weresynthesized on a DNA synthesizer (applied Biosystems Inc., Foster City,Calif.), as described in Example 1. In some ODNs, the normal DNAbackbone phosphodiesterase linkages were replaced with phosphorothioatelinkages, as described in Example 1. To induce degradation of the ODNs,those that did not have an entire phosphorothioate backbone containedphosphorothioate linkages at the 5′ and 3′ ends. Cells were incubatedfor approximately 72 hours with the various ODNs. IL-6 and IFN-γ levelswere determined by ELISA using anti-IL-6 and anti-IFN-γ antibodies, asdescribed in Example 1. The percentages of donors induced by thecombination of different ODNs by at least 3-fold for IL-6 and 5-fold forIFN-γ are set forth for IL-6 levels in Table 9: Percent Induction of anImmune Response (IL-6) and for IFN-γ levels in Table 10: Induction of anImmune Response (IFN-γ).

TABLE 9 Percent Induction of an Immune Response (IL-6) to Multiple ODNsPercent Induction Number of Donors SEQ ID NO: 7 + SEQ ID NO: 1 + SEQ IDNO: 108 100%  4 SEQ ID NO: 7 + SEQ ID NO: 20 + SEQ ID NO: 98 100%  2 SEQID NO: 20 + SEQ ID NO: 108 + SEQ ID NO: 98 100%  2 SEQ ID NO: 7 + SEQ IDNO: 20 + SEQ ID NO: 1 80% 5 SEQ ID NO: 20 + SEQ ID NO: 1 + SEQ ID NO:108 80% 5 SEQ ID NO: 7 + SEQ ID NO: 20 + SEQ ID NO: 108 60% 10 SEQ IDNO: 7 + SEQ ID NO: 108 + SEQ ID NO: 98 38% 16

TABLE 10 Percent Induction of an immune Response (IFN-γ) to MultipleODNs Percent Induction Number of Donors SEQ ID NO: 43 + SEQ ID NO: 40 +SEQ ID NO: 106 100%  5 SEQ ID NO: 32 + SEQ ID NO: 40 + SEQ ID NO: 106100%  5 SEQ ID NO: 43 + SEQ ID NO: 102 + SEQ ID NO: 106 89% 19 SEQ IDNO: 32 + SEQ ID NO: 40 + SEQ ID NO: 42 80% 5 SEQ ID NO: 32 + SEQ ID NO:102 + SEQ ID NO: 106 40% 5 SEQ ID NO: 43 + SEQ ID NO: 40 + SEQ ID NO: 4240% 5

The foregoing data demonstrates the induction of an immune responseafter administration of multiple ODNs including various CpG sequences inhuman PBMC isolated from individual donors. Specifically, these datademonstrate that administration of multiple different ODNssynergistically induces an immune response, as demonstrated by, e.g.,Table 9, in which the percent induction was increased in two of themultiple ODNs to 100%, as measured by IL-6 production. This is alsoshown in Table 10, in which the percent induction was increased to 100%for three of the multiple ODNs, as measured by IFN-γ production.

Example 5

The following example demonstrates in vitro induction of an immuneresponse after administration of a single ODN including multipledifferent CpG sequences. Induction of an immune response was measured byproduction of the cytokines IL-6 and IFN-γ, and cell proliferation inhuman PBMC isolated from individual donors.

Human PBMC were isolated, as described in Example 1. ODNs weresynthesized on a DNA synthesizer (Applied Biosystems Inc., Foster City,Calif.), as described in Example 1. In some ODNs, the normal DNAbackbone phosphodiesterase linkages were replaced with phosphorothioatelinkages, as described in Example 1. To reduce degradation of the ODNs,those that did not have an entire phosphorothioate backbone wereprovided with phosphorothioate linkages at the 5′ and 3′ ends. Cellswere incubated for approximately 72 hours with the various ODNs. IL-6and IFN-γ levels were determined by ELISA using anti-IL-6 and anti-IFN-γantibodies, as described in Example 1. Cell proliferation was determinedby [³H] thymidine incorporation, as described in Example 1. Results areset forth in Table 11: Induction of an Immune Response to a Single ODNIncluding Multiple Different CpG Sequences.

TABLE 11 Induction of an Immune Response to a Single ODN IncludingMultiple Different CpG Sequences IL-6 IFN-γ Proliferation (ELISA)(ELISA) ([³H] T) Donor 1 SEQ ID NO: 1 5 3 18 SEQ ID NO: 43 3 4 3 SEQ IDNO: 1 + SEQ ID NO: 43 23 7 29 Donor 2 SEQ ID NO: 1 5 3 31 SEQ ID NO: 432 4 4 SEQ ID NO: 1 + SEQ ID NO: 43 15 6 57

The foregoing data demonstrates the induction of an immune responseafter administration of a single ODN including multiple different CpGsequences in human PBMC isolated from individual donors. Specifically,these data demonstrate that a single ODN that includes multipledifferent CpG sequences synergistically induces an immune response, suchas is demonstrated by, e.g., an increase in IL-6 levels of 65.2% inDonor 1 and 53.3% in Donor 2 after administration of a single ODN thatincludes SEQ ID NO: 1 and SEQ ID NO: 43, compared to the combined immuneresponse of a single ODN that includes either SEQ ID NO: 1 or SEQ ID NO:43 when administered separately.

Example 6

Over 100 CpG ODNs were then screened for their ability to stimulate PBMCfrom a diverse pool of normal donors (Table 12).

TABLE 12 Characteristics of the PBMC Donor Pool* Characteristic Percentof donor population Sex Male 72 Female 28 Race White 70 Black 28Hispanic 2 Age (years) Range 23-66 Average 40 ± 11 *Characteristics ofthe donor pool from which PBMC samples were randomly derived.

Eleven of the most stimulatory ODNs were then tested for their abilityto stimulate IL6, IgM, Proliferation, or all three responsessimultaneously, on PBMC from over 100 donors (Table 13).

TABLE 13 Oligodeoxynucleotide sequences Sequences of phosphorothioateODNs included in this example. CpG dinucleotides are underlined. K3 isSEQ ID NO: 20, K19 is SEQ ID NO: 7, K23 is SEQ ID NO: 1, K82 is SEQ IDNO: 113, K83 is SEQ ID NO: 114, K84 is SEQ ID NO: 115, K85 is SEQ ID NO:116, K89 is SEQ ID NO: 109, K109 is SEQ ID NO: 117, K110 is SEQ ID NO:98, K123 is SEQ ID NO: 108, and K121 is SEQ ID NO: 112. Name Sequence*CpG ODN (K type) K3 A T C G A C T C T C G A G C G T T C T C K19       AC T C T C G A G C G T T C T C K23               T C G A G C G T T C T CK82       A C T C T G G A G C G T T C T C K83       A C T C T C G A G GG T T C T C K84       A C T C T C G A G C G T T C T A K85       C A T CT C G A G C G T T C T C K89           A C T C T T T C G T T C T C K109              T C G A G C G T T C T K110               T C G A G G C T TC T C K123               T C G T T C G T T C T C Control ODN K121      A C T C T T G A G T G T T C T C *The CpG motif is underlined ineach ODN.

ODNs K3, K19 and K23 were the most effective at inducing proliferation,stimulating significant (>5-fold) proliferation by >90% of donor samples(Table 14).

TABLE 14 Percent of Donor Samples Responding to Stimulation byIndividual ODN IL-6 IgM ODN Proliferation Production Production ALL K393 (54) 82 (50) 59 (49) 62 (38) K19 92 (76) 52 (61) 88 (56) 48 (42) K2393 (44) 71 (45) 55 (33) 64 (28) K82 88 (8) 56 (9) 89 (9) 63 (8) K83 63(8) 67 (9) 78 (9) 63 (8) K84 67 (9) 73 (11) 89 (9) 63 (8) K85 56 (9) 73(11) 89 (9) 60 (8) K89 57 (7) 67 (9) 57 (7) 50 (6) K109 67 (9) 64 (11)71 (7) 57 (7) K110 81 (68) 36 (58) 60 (53) 27 (41) K123 90 (70) 52 (54)65 (55) 43 (46) K121(nonCG) 21 (68) 11 (47) 35 (49)  3 (39) *5 × 10⁵freshly isolated PBMC (N = 7-76) were stimulated with 1 μM of CpG ODNfor 72 hours IL-6 and IgM levels in culture supernatants were determinedby ELISA, while proliferation was measured by ³H-thymidineincorporation. The percent of donor samples with significantly elevatedresponses to each ODN(exceeding media alone by >5-fold) is shown. Thenumber of samples tested for each condition is indicated in parentheses.The percent of subjects responding (>5-fold) in all 3 assays (ALL, N =6-46) is provided in the final column. Not all PBMC samples were testedin all assays, and these were not included in the “ALL” analysis.

Of note, each of these ODNs contains at least two different CpG motifs(including CTCGAG and AGCGTT). By comparison, ODNs K83 and K89 (whichexpress a single CpG motif) failed to trigger over a third of the PBMCsamples to proliferate. Control ODN stimulated a response in only 20% ofthe donor samples and the magnitude of the response was significantlylower (p<0.05) compared to CpG ODNs (Table 14). None of the CpG ODNsstudied was able to stimulate cells from every donor to proliferate.

IL-6 and IgM production were also analyzed. Greater than 80% of donorsamples were stimulated to secrete IL-6 by ODN K3 (Table 14), which, asnoted above, expresses multiple different CpG motifs. By comparison,ODNs expressing a single motif (even if that motif was present inmultiple copies) were less active (i.e. K82, K89, K110 and K123). Therewas no discernable pattern for IgM secretion with respect to the lengthor number of CpG motifs contained in an ODN, however thephosphorothioate backbone contributes considerably to IgM inductionsince many individuals responded to the non-CpG ODN (Table 14).Importantly, there was some bias in the type of stimulation elicited bycertain ODNs. For example, K110 was one of the strongest inducers ofproliferation but was relatively weak at stimulating IgM or IL-6secretion, whereas K3 was a potent inducer of proliferation and IL-6production, but not IgM secretion (Table 14).

To examine the breadth of the stimulatory response elicited byindividual ODN, their ability to simultaneously trigger at least a 5fold increase in the response for three exemplary immune parameters(proliferation, IgM and IL-6 production) was examined. Whereas some ODNswere very effective at inducing one or two responses, no single ODNelicited all three types of response in greater than 64% of the donorpool (Table 14). For example, K3 stimulated 92% of the PBMC samples toproliferate and 82% to produce IL-6, but triggered only 59% of the samedonors to produce IgM (Table 14). Of note, the control ODN elicited acomprehensive response in only 3% of the subjects (Table 14).

To determine whether assay variability contributed to thisheterogeneity, the response of freshly isolated PBMC from the samedonors was monitored on multiple occasions. An individual's pattern ofreactivity to each ODN was quite reproducible, indicating that theobserved heterogeneity was not due to assay variability.

Several novel insights are provided by this series of studies: 1)Although PBMC from all donors respond to at least one member of the ODNpanel, no single ODN activated PBMC from all donors in all assays. 2)Different ODNs were frequently required for the induction of optimalIL-6, IgM and proliferative responses by the same subject. 3) ODNscontaining multiple CpG motifs were generally more active than thosecontaining a single motif for stimulating proliferation and IL-6production.

Example 7

This example demonstrates that ODN mixtures are broadlyimmunostimulatory.

To overcome the limitations associated with the use of individual ODNs,mixtures were prepared from ODNs selected for their strong activity onPBMC from different subjects in different assays. Preliminary studies(see above) showed that mixtures that included three ODNs were optimallyeffective. Several such mixtures stimulated a significantly broader poolof donor PBMC in more assays than any single ODN (Table 15).

TABLE 15 Percent of Donor Samples Responding to Stimulation by K typeODN Mixtures* ODN Mixture Proliferation IL-6 IgM ALL K3 K19 K110 96 (25)95 (19) 91 (23) 94 (17) K19 K23 K123 100 (17)  86 (14) 100 (14)  83 (12)K3 K110 K123 94 (17) 93 (15) 87 (15) 77 (13) K3 K23 K123 95 (20) 78 (18)89 (18) 67 (15) K3 K19 K123 95 (20) 67 (18) 83 (18) 53 (15) K19 K110K123 77 (31) 50 (20) 82 (22) 35 (17) *5 × 10⁵ freshly isolated PBMC (N =12-31) were stimulated with mixtures of three ODNs (0.33 μM of eachcomponent to yield a final concentration of 1 μM for the mixture) for 72hours. The percent of donor samples responding by proliferating orproducing IL-6, IgM or all three responses (ALL, N = 12-17) wasdetermined as described in the legend to Table 3. The number of samplestested for each condition is shown in parentheses.

The most effective mixture (composed of 0.33 μM each of K3, K19 and K110yielding a composite ODN concentration of 1 μM) stimulated 94% of donorsto proliferate and secrete IgM and IL-6. This significantly exceeded thebreadth of the activity elicited by any single ODN at a concentration of1 μM (p<0.05). Thus, this mixture of ODNs exceeded the ability ofindividual ODN to trigger a diverse population of donors to mount abroad range of immune responses.

To examine the magnitude of the stimulation elicited by this mixture,the response induced by K3/K19/K110 was compared to each of itscomponents on 11 donors. In this study, individual ODNs were used at aconcentration of 1 μM while each component of the mixture was used at0.33 μm, yielding a final concentration of 1 μM. On average, the mixturestimulated a 13-fold increase in proliferation, a 6-fold increase inIL-6 production, and an 11-fold increase in IgM secretion. The responseto the mixture did not differ significantly from the stimulation inducedby the most active single ODN in each of these assays.

Murine studies suggest that CpG ODNs are useful as immunotherapeuticagents. However, ODNs that are highly active in mice are poorlyimmunostimulatory in man. Considerable effort has been invested inidentifying ODNs capable of activating human cells. The described CpGODNs vary dramatically in sequence, structure, as well as CpG number andplacement. No studies thus far have specifically addressed heterogeneityin the human response to CpG ODNs. In this example, 11 highly activeODNs selected from a panel of >100 sequences were used to stimulate IgM,IL-6 and proliferative responses in PBMC from a large and diverse panelof normal donors. Results indicate that the human response to CpG ODNsis heterogeneous. No single ODN was stimulatory in all donors, andoptimal Ig, cytokine and proliferative responses were commonly elicitedby different ODNs, even in the same donor. A mixture was identified thatinduced a broader response in a greater fraction of donors than anysingle ODN.

Without being bound by theory, there are several potential sources forthis heterogeneity. Since CpG ODNs trigger monocytes to secrete IL-6, Bcells to secrete IgM, and multiple cell types to proliferate,heterogeneity between individuals may reflect differences in therelative frequency of each cell type as well as the reactivity of eachcell population to specific CpG motifs. That is, if B cells respondbetter to K3 than K19, subjects who have higher number of B cells mightshow a stronger response to K3. Additional variability may arise fromprevious exposure to CpG DNA from environmental sources (such aspathogens or vaccines)—exposures that vary between individuals and mightalter their subsequent response to specific ODNs.

Without being bound by theory, superimposed on this subject-to-subjectvariability may be population-based differences in CpG recognition. Forexample, pathogens endemic to distinct geographic locations may expressdifferent CpG motifs, and thus exert selective pressure leading todivergence in CpG recognition by inhabitants of those locations.Consistent with such a possibility is the finding that CpG recognitionby rodents and primates has diverged over evolutionary periods.

It should be noted that the heterogeneity in the response was not due tointer assay variation since the individual pattern of response to CpGODNs was reproducible in subjects tested on multiple occasions with thesame sequences. Care was also taken to minimize the effect of sequenceindependent stimulation associated with the use of phosphorothioateODNs. Specifically, ODNs were studied at the low fixed concentration of1 μM, and responses were considered positive only if they exceededbackground by >5-fold (since control ODN rarely induced such strongnon-specific stimulation, FIG. 1).

A critical finding from the current work is that a mixture of ODNs ismore broadly active than any single ODN. Each component of the optimallystimulatory mixture identified in this study expressed a different CpGmotif at its 5′ end—the site of greatest immunostimulatory activity.Apparently, without being bound by theory, PBMC recognize and respond tomotifs that are stimulatory in that individual without interference bysequences that are less active.

Example 8

The response to D ODN was also investigated. Briefly, mononuclear cellswere isolated from normal donors by density gradient centrifugation overFicoll-Hypaque. Cells were cultured for 72 hours in RPMI supplementedwith 10% heat inactivated FCS, 100 U/ml penicillin, 100 μg/mlstreptomycin, and 2 mM L-glutamine. ODNs were synthesized at the CBERCore Facility.

D19* GGtgcatcgatgcagGGGGG (SEQ ID NO: 32) D29* GGtgcaccggtgcagGGGGG (SEQID NO: 42) D113* GGtgcatgcatacagGGGGG (SEQ ID NO: 106) D35*GGtgcatcgatgcaggggGG (SEQ ID NO: 32) D28* GGtgcgtcgatgcagGGGGG (SEQ IDNO: 40) D103* GGtcaccgtggcagGGGGG (SEQ ID NO: 118) D106*GGtgtgtcgatgcagGGGGG (SEQ ID NO: 102) D122* GGtgcattgatgcagGGGGG (SEQ IDNO: 107) AA3M* GGgcatgcatgGGGGG (SEQ ID NO: 119) *bases shown in capitalletters are phosphorothioate while those in lower case arephosphodiester

All ODN contained less than 0.1 EU/mg of endotoxin as measured by theLimulus amoebocyte lysate. 5×10⁵ PBMC were stimulated in vitro for 72hours with 1 μM ODN. Expression of Interferon gamma was then assessedusing standard methods. For example, culture supernatants were detectedby ELISA (For example, see Gold Book of Immunological Assays, of Harlowand Lane, “Antibodies, a Laboratory Mannual,” Cold Spring HarborLaboratory, New York, 1988). ELISA results were quantitated usingstandard curves generated using recombinant IFN-γ. The limit ofdetection of the assays was 5-20 pg/ml. In those cases where interferonproduction was below assay sensitivity, the lower limit of detection wasused to calculate the stimulation index. All assays were performed intriplicate. ODNs were considered stimulatory if the response theyinduced exceeded that of unstimulated PBMC from that subject by >5-fold.This minimized the effect of sequence-independent background stimulationassociated with the use of phosphorothioate ODN.

TABLE 16 IFN-γ response of normal donors to a panel of D ODN ODN %responders N D19 93 42 D29 91 23 D113 87 23 D35 82 17 D28 79 19 D103 5812 D106 57 28 D122 10 31 AA3M 11 19

None of the D type ODN including a CpG motif stimulated all of thesubjects. However, some of the D type ODN stimulated a plurality ofsubjects.

Combinations of D type ODN were then assessed. The results are shown inTable 17.

TABLE 17 Effect of using mixtures of D ODN on IFN-γ response D-Mixture %responders N = D19 D28 D113 100 5 D35 D28 D113 100 5 D19 D106 D113 89 19D35 D28 D29 80 5 D35 D106 D113 40 5 D19 D28 D29 40 5Thus, a composition including D19, D28, and D113, a compositionincluding D35, D28, and D113, a composition including D19, D106, andD113, and a composition including D35, D28, and D29 induced an immuneresponse in 80% or more of the subjects in the population.

It will be apparent that the precise details of the methods orcompositions described may be varied or modified without departing fromthe spirit of the described invention. We claim all such modificationsand variations that fall within the scope and spirit of the claimsbelow.

1. An oligodeoxynucleotide composition comprising a firstoligodeoxynucleotide of 20 to 50 nucleotides in length, wherein thefirst oligodeoxynucleotide comprises the nucleic acid sequence set forthas SEQ ID NO: 106; and a second oligodeoxynucleotide of 16 to 50nucleotides in length, wherein the second oligodeoxynucleotide comprisesan unmethylated CpG motif, and wherein the second oligodeoxynucleotidecomprises a sequence represented by the formula 5′ X₁X₂X₃ Pu₁ Py₂ CpGPu₃ Py₄ X₄X₅X₆(W)_(M)(G)_(N)-3′, wherein the central CpG motif isunmethylated, Pu is a purine nucleotide, Py is a pyrimidine nucleotide,X and W are any nucleotide, M is any integer from 0 to 10, and N is anyinteger from 4 to 10; wherein the first and the secondoligodeoxynucleotides differ in their nucleic acid sequence and whereinthe composition induces an immune response.
 2. The oligodeoxynucleotidecomposition of claim 1, wherein the first oligodeoxynucleotide comprisesmultiple CpG motifs.
 3. The oligodeoxynucleotide composition of claim 1,wherein the second oligodeoxynucleotide comprises a single CpG motif. 4.The oligodeoxynucleotide composition of claim 1, wherein the secondoligodeoxynucleotide comprises the nucleic acid sequence set forth asSEQ ID NO: 32, SEQ ID NO: 102 or SEQ ID NO:
 42. 5. Theoligodeoxynucleotide composition of claim 1, further comprising a thirdoligodeoxynucleotide having a different nucleic acid sequence than thefirst oligodeoxynucleotide and a different sequence than the secondoligodeoxynucleotide, wherein the third oligodeoxynucleotide is 16 to 50nucleotides in length, wherein the third oligodeoxynucleotide comprisesan unmethylated CpG motif, and wherein the second oligodeoxynucleotidecomprises a sequence represented by the formula 5′ X₁X₂X₃ Pu₁ Py₂ CpGPu₃ Py₄ X₄X₅X₆(W)_(M) (G)_(N)-3′, wherein the central CpG motif isunmethylated, Pu is a purine nucleotide, Py is a pyrimidine nucleotide,X and W are any nucleotide, M is any integer from 0 to 10, and N is anyinteger from 4 to
 10. 6. The oligodeoxynucleotide composition of claim1, wherein the second oligodeoxynucleotide comprises the nucleic acidsequence set forth as SEQ ID NO:
 32. 7. The oligodeoxynucleotidecomposition of claim 1, wherein the second oligodeoxynucleotidecomprises the nucleic acid sequence set forth as SEQ ID NO:
 102. 8. Theoligodeoxynucleotide composition of claim 1, wherein the firstoligodeoxynucleotide, the second oligodeoxynucleotide or both comprisephosphodiester bases.
 9. The oligodeoxynucleotide composition of claim1, wherein the first oligodeoxynucleotide, the secondoligodeoxynucleotide or both comprise one or more phosphothioate bases.10. The oligodeoxynucleotide composition of claim 1, wherein the firstoligodeoxynucleotide, the second oligodoexynucleotide or both aremodified to prevent degradation.
 11. The oligodeoxynucleotidecomposition of claim 1, wherein the first oligodeoxynucleotide, thesecond oligodeoxynucleotide or both comprise a targeting moiety.
 12. Theoligodeoxynucleotide composition of claim 11, wherein the targetingmoiety is selected from the group consisting of a cholesterol, avirosome, a liposome, a lipid, and a target cell specific binding agent.13. The oligodeoxynucleotide composition of claim 5, wherein the thirdoligodeoxynucleotide comprises phosphodiester bases.
 14. Theoligodeoxynucleotide composition of claim 5, wherein the thirdoligodeoxynucleotide comprises one or more phosphothioate bases.
 15. Theoligodeoxynucleotide composition of claim 5, wherein the thirdoligodeoxynucleotide is modified to prevent degradation.
 16. Theoligodeoxynucleotide composition of claim 5, wherein the firstoligodeoxynucleotide, the second oligodeoxynucleotide, the thirdoligodeoxynucleotide or any combination thereof comprise a targetingmoiety.
 17. The oligodeoxynucleotide composition of claim 16, whereinthe targeting moiety is selected from the group consisting of acholesterol, a virosome, a liposome, a lipid, and a target cell specificbinding agent.
 18. The oligodeoxynucleotide composition of claim 1,comprising one of: a) an oligodeoxynucleotide comprising a sequence asset forth as SEQ ID NO: 32, an oligodeoxynucleotide comprising thesequence as set forth as SEQ ID NO: 40, and an oligodeoxynucleotidecomprising the sequence as set forth as SEQ ID NO: 106; or b) anoligodeoxynucleotide comprising the sequence as set forth as SEQ ID NO:32, an oligodeoxynucleotide comprising the sequence as set forth as SEQID NO: 102, and an oligodeoxynucleotide comprising the sequence as setforth as SEQ ID NO:
 106. 19. A pharmaceutical composition comprising atherapeutically effective amount of the oligodeoxynucleotide compositionof claim 1 in a pharmaceutically acceptable carrier.
 20. Thepharmaceutical composition of claim 19, further comprising atherapeutically effective amount of a third oligodeoxynucleotide havinga different nucleic acid sequence than the first oligodeoxynucleotideand a different sequence than the second oligodeoxynucleotide.
 21. Thepharmaceutical composition of claim 20, wherein the thirdoligodeoxynucleotide is 20 to 50 nucleotides in length, wherein thethird oligodeoxynucleotide comprises an unmethylated CpG motif, andwherein the third oligodeoxynucleotide comprises a sequence representedby the formula 5′ X₁X₂X₃ Pu₁ Py₂ CpG Pu₃ Py₄ X₄X₅X₆(W)_(M) (G)_(N)-3′,wherein the central CpG motif is unmethylated, Pu is a purinenucleotide, Py is a pyrimidine nucleotide, X and W are any nucleotide, Mis any integer from 0 to 10, and N is any integer from 4 to 10, whereinthe third oligodeoxynucleotide comprises the nucleic acid sequence setforth as SEQ ID NO:
 32. 22. The pharmaceutical composition of claim 20,wherein the third oligodeoxynucleotide is 20 to 50 nucleotides inlength, wherein the third oligodeoxynucleotide comprises an unmethylatedCpG motif, and wherein the second oligodeoxynucleotide comprises asequence represented by the formula 5′ X₁X₂X₃ Pu₁ Py₂ CpG Pu₃ Py₄X₄X₅X₆(W)_(M) (G)_(N)-3′, wherein the central CpG motif is unmethylated,Pu is a purine nucleotide, Py is a pyrimidine nucleotide, X and W areany nucleotide, M is any integer from 0 to 10, and N is any integer from4 to 10, wherein the third oligodeoxynucleotide comprises the nucleicacid sequence set forth as SEQ ID NO:
 102. 23. The pharmaceuticalcomposition of claim 20, wherein the third oligodeoxynucleotide is 16 to50 nucleotides in length, wherein the third oligodeoxynucleotidecomprises an unmethylated CpG motif, and wherein the secondoligodeoxynucleotide comprises a sequence represented by the formula 5′X₁X₂X₃ Pu₁ Py₂ CpG Pu₃ Py₄ X₄X₅X₆(W)_(M) (G)_(N)-3′, wherein the centralCpG motif is unmethylated, Pu is a purine nucleotide, Py is a pyrimidinenucleotide, X and W are any nucleotide, M is any integer from 0 to 10,and N is any integer from 4 to 10, wherein the thirdoligodeoxynucleotide comprises the nucleic acid sequence set forth asSEQ ID NO:
 40. 24. A method for stimulating an immune response in asubject, comprising administering to the subject a therapeuticallyeffective amount of the oligodeoxynucleotide composition of claim 1,thereby stimulating the immune response, wherein the immune response issubstantially stimulated as compared to administration of the firstoligodeoxynucleotide.
 25. The method of claim 24, further comprisingadministering to the subject a therapeutically effective amount of athird oligodeoxynucleotide having a different nucleic acid sequence thanthe first oligodeoxynucleotide and a different sequence than the secondoligodeoxynucleotide, wherein the third oligodeoxynucleotide is 16 to 50nucleotides in length, wherein the third oligodeoxynucleotide comprisesan unmethylated CpG motif, and wherein the third oligodeoxynucleotidecomprises a sequence represented by the formula 5′ X₁X₂X₃ Pu₁ Py₂ CpGPu₃ Py₄ X₄X₅X₆(W)_(M)(G)_(N)-3′, wherein the central CpG motif isunmethylated, Pu is a purine nucleotide, Py is a pyrimidine nucleotide,X and W are any nucleotide, M is any integer from 0 to 10, and N is anyinteger from 4 to
 10. 26. The method of claim 24, wherein the secondoligodeoxynucleotide comprises the nucleic acid sequence set forth asSEQ ID NO:
 102. 27. The method of claim 25, wherein the secondoligodeoxynucleotide comprises the nucleic acid sequence set forth asSEQ ID NO:
 32. 28. The method of claim 25, wherein the secondoligodeoxynucleotide comprises the nucleic acid sequence set forth asSEQ ID NO:
 40. 29. The method of claim 24, wherein the immune responsecomprises the production of interferon gamma.
 30. A method forstimulating an immune response in a subject, comprising administering tothe subject a therapeutically effective amount of theoligodeoxynucleotide composition of claim 18, thereby stimulating theimmune response.
 31. The method of claim 30, wherein the immune responsecomprises the production of interferon gamma.
 32. An isolatedoligodeoxynucleotide of 20 to 50 nucleotides in length, wherein theisolated oligodeoxynucleotide comprises the nucleic acid sequence setforth as SEQ ID NO:
 106. 33. A composition comprising the isolatedoligonucleotide of claim 32 in a pharmaceutically acceptable carrier.34. A method for stimulating an immune response in a subject, comprisingadministering to the subject a therapeutically effective amount of theoligodeoxynucleotide composition of claim 5, thereby stimulating theimmune response.
 35. The method of claim 34, wherein the immune responsecomprises the production of interferon gamma.
 36. Theoligodeoxynucleotide composition of claim 4, wherein the secondoligodeoxynucleotide comprises the nucleic acid sequence set forth asSEQ ID NO: 40.